The production of industrial enzymes by the fermentation of microorganisms, whether they be fungi or bacteria, in an aqueous nutrient media is well-known. Depending on the nature of the particular microorganism, the enzyme(s) produced may be intracellular or extracellular. Various microorganisms produce two or more useful enzymes during fermentation. When one of the enzymes is intracellular and one is extracellular, enzyme separation is accomplished by separating the enzyme containing cells from the fermentation broth and recovering the intracellular enzyme. The extracellular enzyme can be recovered from the broth by conventional methods. An example of this type of separation is illustrated by co-pending application Ser. No. 425,713 where there is described the fermentation of an organism of the species F. arborescens to produce intracellular glucose isomerase and extracellular protease and separation of the enzymes by centrifugation of the glucose isomerase containing cells with recovery of the protease containing supernatant.
In some instances, such as where the enzyme must be solubilized by use of a detergent or by rupturing the cell walls, the separation of the solubilized enzyme from the cells or cell fragments can be problematical. This type of separation difficulty is described in U.S. Pat. No. 4,144,130 where it is pointed out that when the pullulanase produced by Klebsiella pneumosiae is removed from intact cells with detergents, separation of the cells from the supernatant by centrifugation requires uneconomically high g-numbers and separation by centrifugation is difficult because of the presence of the detergent. This patent discloses a method of separating the enzyme from the cells or cell fragments which involves feeding the fermentation broth into an aqueous solution of a low molecular weight, substituted or unsubstituted polyalcohol, polyether, polyester, polyvinylpyrrolidone or polysaccharide and an inorganic salt or a solution containing two of the high molecular weight compounds and separating the phases from each other which results in the cells or cell fragments being retained in one phase and the enzyme in the other. These patentees who also describe their work in Biotechnology and Bioengineering, Vol. XX, pages 1989-2005 (1978) wherein they disclose the use of dextran as a species of polysaccharide useful in this process.
A method for recovering phospholipase A from Escherichia coli in which the cells are homogenized, the cell liquid is separated by centrifugation and the enzyme is dissolved from the cell membrane all in a phase-distribution multi-phase system containing such immiscible polymers as polyethyleneglycol and dextran is disclosed in Biochemistry, Vol. 12, No. 13, 1973.
In certain fermentations, two useful, extra-cellular enzymes are produced, and since both enzymes are freely dispersed in the fermentation broth even after removal of the cells, their separation is difficult. An example of such a system is the fermentation in an aqueous nutrient medium of Bacillus amyloliquefacieus in which extracellular protease and amylase are produced. These enzymes are difficult to separate and it is an object of the present invention to provide a novel method for their separation.